Acetaminophen (APAP) is the most commonly used medication in the United States but is also the most frequent cause of drug-induced hepatotoxicity. The long-term objective of this research is to elucidate the pharmacogenetic mechanisms that contribute to variability in individual risk for APAP-induced hepatotoxicity. Our working hypothesis is that genetic polymorphisms that modify the expression and function of enzymes and regulatory proteins involved in APAP toxification and detoxification can be used to identify individuals that have increased risk for APAP hepatotoxicity. The major focus of this work are genes encoding the enzymes that have been identified as critical to these pathways, including the UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and cytochromes P450 (CYPs). In preliminary work, we established UGT1A1, 1A6, and 1A9 as major APAP glucuronidation enzymes, and identified 3 linked amino acid polymorphisms in the UGT1A6 gene that alter APAP glucuronidation by recombinant enzyme, and also 3 linked SNPs in the 3'UTR region shared by all UGT1A isoforms that were associated with higher APAP glucuronidation in a human liver bank. Consequently, in Aim 1 we propose to elucidate the molecular mechanisms underlying the effects of the UGT1A6 cSNPs and the UGT1A 3'-UTR SNPs on APAP glucuronidation through studies of protein stability/localization, mRNA stability, and translation efficiency. In Aim 2 we will determine the effect of these UGT polymorphisms and other known functional polymorphisms in the SULT and CYP genes on rates of APAP glucuronidation, sulfation, and oxidation measured in volunteers that receive a therapeutic dose of APAP. We will also determine whether metabolism of APAP is different in African-Americans compared with European-Americans (a novel and untested hypothesis). In Aim 3 we will utilize DNA samples from 2 ongoing studies of APAP-induced hepatotoxicity to determine whether this validated subset of UGT, CYP, and SULT polymorphisms can be used to identify patients predisposed to developing hepatotoxicity resulting from APAP use (overdose or unintentional toxicity). Public health relevance: Our goal is to identify of a set of genetic markers that could be used by physicians and patients to guide their choice of safe and effective analgesic drugs, and identify patients admitted to an emergency room at high risk for hepatotoxicity (requiring special treatment) following an APAP overdose.